buffalo cheese whey proteins identification

RESEARCH ARTICLE

Buffalo Cheese Whey Proteins, Identification
of a 24 kDa Protein and Characterization of
Their Hydrolysates: In Vitro Gastrointestinal
Digestion
Juliana C. Bassan1, Antonio J. Goulart1, Ana L. M. Nasser1, Thaís M. S. Bezerra2, Saulo
S. Garrido2, Cynthia B. Rustiguel3, Luis H. S. Guimarães3, Rubens Monti1*
1 Faculdade de Ciências Farmacêuticas, UNESP Univ EstadualPaulista, Departamento de Alimentos e
Nutrição, Araraquara - SP, Brazil., 2 Instituto de Química, UNESP Univ EstadualPaulista, Departamento de
Bioquímica e Química Tecnológica, Araraquara - SP, Brazil., 3 Universidade de São Paulo, Departamento
de Biologia, Ribeirão Preto - SP, Brazil
* montiru@fcfar.unesp.br

OPEN ACCESS
Citation: Bassan JC, Goulart AJ, Nasser ALM,
Bezerra TMS, Garrido SS, Rustiguel CB, et al. (2015)
Buffalo Cheese Whey Proteins, Identification of a 24
kDa Protein and Characterization of Their
Hydrolysates: In Vitro Gastrointestinal Digestion.
PLoS ONE 10(10): e0139550. doi:10.1371/journal.

pone.0139550
Editor: Sabato D'Auria, CNR, ITALY
Received: March 11, 2015
Accepted: September 14, 2015
Published: October 14, 2015
Copyright: © 2015 Bassan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
available from Figshare: http://dx.doi.org/10.6084/m9.
figshare.1482092.
Funding: Financial and research support were
provided by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Fundação de
Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (J. C. Bassan), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) (T. M. S. Bezerra) and Programa Nacional
de Pós-Doutorado (PNPD-CAPES) (A. J. Goulart).
Rubens Monti is a Research Fellow of CNPq. The

Abstract
Milk whey proteins are well known for their high biological value and versatile functional
properties, characteristics that allow its wide use in the food and pharmaceutical industries.
In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins
present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used
the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total
antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of
Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for
H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared
with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion,
several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

Introduction
Cheese whey, which is a byproduct of cheese production, is a greenish-yellow solution composed of water, lactose, proteins and minerals [1–2] that represents 85–90% of the milk volume
[3]. Dairy experts from around the world have sought to use whey because it contains highly
nutritious components that should not be wasted and because it is a potent environmental

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Buffalo Cheese Whey Hydrolysates Production

funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

pollutant [4]. Milk whey proteins are well known for their high biological value and versatile
functional properties, characteristics that allow its wide use in the food [5] and pharmaceutical
[6] industries. These compact and globular proteins are responsible for 20% of the total protein
contained in milk and, unlike casein, remain soluble at pH 4.6 [7]. The main proteins present
in cheese whey are β-lactoglobulin (3.2 g.L-1, 18.3 kDa), α-lactalbumin (1.2 g.L-1, 14.2 kDa),
serum albumin (0.4 g.L-1, 66.0 kDa), immunoglobulin (0.8 g.L-1, 146–1030 kDa), and lactoferrin (0.2 g. L-1, 80 kDa), among others [2,8]. Current technologies allow the separation, isolation
and purification of proteins from whey, usually through a combination of methods, such as filtration techniques associated with chromatography [5]. A wide variety of products, such as

concentrates (35–80% protein), protein isolates (minimum protein content of 90%), protein
fractions (α-lactalbumin, β-lactoglobulin and lactoferrin) and hydrolysates [4], which are classified as GRAS for use in food products [9,4], are available on the market. The physical, chemical and functional properties of macromolecules such as proteins can be modified and
improved by enzymatic hydrolysis, particularly the absorption capacity, without affecting their
nutritional value [10], while reducing allergenic processes [11]. Protein hydrolysates of cheese
whey are a source of bioactive peptides [12] with opioid, antihypertensive, antithrombotic,
antioxidant, immunomodulatory and antimicrobial activities [13–17] and of essential amino
acids [18–19], which are often used as protein supplementation for infants and athletes and for
parenteral administration [20]. There are few reports in the literature concerning bioactive
peptides obtained by the hydrolysis of whey proteins, suggesting that further studies are
required to fully understand this byproduct [21].
Genetic variants have being identified in buffaloes that are present in different fractions of
milk proteins, affecting the composition and technological properties of these fractions [22]. In
addition, studies performed in different parts of the world show that different buffalo breeds
present both variants of α-lactalbumin and β-lactoglobulin [23–24]. Although bovine cheese
whey proteins have been extensively studied, studies regarding buffalo milk proteins are quite
scarce [25].
In this article, we present a methodology to hydrolyze the proteins from Murrah buffalo
cheese whey, and a study of the hydrolysis products. We also describe the amino acid sequencing of a discovered variant protein of β-lactoglobulin.

Materials and Methods

In all cases, the experiments were performed in triplicate, and the experimental error was never
greater than 5%.

Reagents
Pepsin, trypsin, chymotrypsin, carboxypeptidase-A, bile salts, pancreatin, (±)-6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2 diphenyl-1-picrylhydrazyl
(DPPH) were purchased from Sigma-Aldrich Co. Renin from Aspergillus niger var. awamori
(Chr. Hansen Ind. Com. Ltd) was acquired in the local market. Other reagents were of analytical grade.

The productin of buffalo cheese whey
The buffalo cheese whey was prepared by adding 0.6% (v/v) renin from A. niger and 0.5% (v/v)
0.5 mol. L-1 CaCl2 to buffalo milk at 35°C for 1 h. After casein precipitation, the whey was filtered in gauze [26] and dialyzed in cellulose membrane (12 kDa, Sigma) under constant magnetic stirring at 8°C. Periodic water exchange for lactose removal was also performed [27]. Fat

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Buffalo Cheese Whey Hydrolysates Production

was removed by adsorption in kaolin (20 g. L-1 w/v), with subsequent centrifugation at 7400 x
g at 4°C for 30 min. The treated whey was stored at -18°C until further use.

Total protein, lactose and fat determination
The concentrations of protein, lactose and fat were determined following the methodologies of
Bradford [28], Miller [29] and Gerber [30], respectively. Absorptivities were 0.0208 (μg prot.
mL-1) -1. cm-1 and 247.23 mol. L-1. cm-1, using bovine serum albumin (BSA) and lactose as
standards, respectively.

PAGE and SDS-PAGE analysis of buffalo chees whey proteins
Ten percent PAGE [31] and 12% SDS-PAGE [32] analyses were performed to obtain the whey
proteins profiles and to monitor and analyze the hydrolysis of these proteins. Protein bands
from PAGE and from SDS-PAGE were stained with silver [33] and with Brilliant Blue G-Colloidal (Sigma), respectively. For the SDS-PAGE analysis, a GE Healthcare Life Sciences molar
mass standard composed of phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and α-lactalbumin
(14.4 kDa) was used.

Molar mass determination of the variant protein from the buffalo cheese
whey
The molar mass of the new protein was determined from the SDS-PAGE (Fig 1A and 1B)
using the Least Square Method, according to the formula:
3
2

3
2
n
n
n
X
X
X
2
6
f ðx Þx 7
xi 7 " # 6
x
6 k¼1 i i 7
7
6 k¼1 i
a1
k¼1
7
6

7
6
¼6 n
7
7
6 n
X
6
6X
7
7
a
2
4
5
4
f ðxi Þ 5
xi
n
k¼1

k¼1

Where:
xi: Rf value
f(xi): The Molar Mass log
a1: Constant to be found
a2: Constant to be found
n: Number of elements (proteins).
Cited constants above are the function constants g(x) = a1x + a2, there is, the function
modeling the experiment.

Isolation and characterization of the 24 kDa protein from the buffalo
cheese whey
Proteins were 10-fold concentrated by lyophilization and submitted to 12% SDS-PAGE as previously described. After the running, the gel was processed to visualize the proteins of interest
and to serve as a guide for cutting slices containing the 24 kDa protein.
The gel slices were dried and submitted to digestion using 0.5 μg of trypsin (Promega) in 0.1
M sodium bicarbonate buffer, pH 8.0. After digestion, the peptides were loaded in a Poros 50
R2 reverse phase column (Perseptive Biosystems). The purified peptides were added with
matrix solution (50 mg. mL-1 α-ciano-4-hidroxycinnamic acid, 50% acetonitrile and 0.1% trifluoroacetic acid), and 2 μL samples were used for MALDI-TOF/TOF-MS analysis. The MS/

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Buffalo Cheese Whey Hydrolysates Production

Fig 1. Electrophoretic profile of the buffalo cheese whey. a) Ten percent PAGE silver stained protein
profile of treated bovine (1) and buffalo (2) whey. b) Twelve percent SDS-PAGE of the bovine and buffalo milk
whey proteins. (1) Molar mass standards; (2) bovine milk whey; (3) buffalo milk whey. Ig: immunoglobulin;
BSA: bovine serum albumin; α-La: alpha-lactalbumin; β-Lg: beta-lactoglobulin.
doi:10.1371/journal.pone.0139550.g001

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Buffalo Cheese Whey Hydrolysates Production

MS profiles were analyzed using the MASCOT software (Matrix Science, London, UK) and the

Swiss Prot and NCBInr databases.

Enzymatic hydrolysis of cheese whey proteins
After removing the fat and lactose, the whey (10 mL) was hydrolyzed with pepsin (429 U, pH
3.0; 50°C), trypsin (432 U), chymotrypsin (18.3 U) and carboxypeptidase-A (16.7 U). Trypsin,
chymotrypsin and carboxypeptidase-A were added to the whey at 50°C and pH 9.5 by two different methods (M1 and M2). In the M1, the enzymes were simultaneously added, and the
time of hydrolysis was 180 min, producing the H1 hydrolysate, whereas in the M2, these same
enzymes were added to the whey with a 10 min interval between each addition, at the same pH
and temperature, with a 24 hours reaction, producing the H2 hydrolysate. The end of the
hydrolysis was determined by SDS-PAGE and by HPLC analysis, performed with samples collected at different reaction times and that were incubated in a boiling bath to denature the
enzymes used.

Antioxidant activity of the buffalo cheese whey hydrolysates
Briefly, 1 mL mmol. L-1 DPPH• solution in methanol 80% was mixed with 0.5 mL of samples
and standard. The mixture was kept at room temperature for 30 min; then the absorbance at
517 nm was measured in a BioTek Synergy H1 reader (BioTek Instruments, USA). Trolox (0–
150 μmol. L-1) was used as standard. The results were expressed as a Trolox Equivalent (TE)
per μmol. L-1 [34].

Quantification of the degree of hydrolysis (DH) using OPA and TNBS
The OPA assay was carried out with a modification of the Spellman et al. method [35], adding
0.02 mL of sample or standard to 2.4 mL of OPA/NAC reagent without SDS (the modification
of the method). The OPA/NAC reagent was prepared mixing 10 mL OPA (50 mmol. L-1), 10
mL NAC (50 mmol. L-1) and 80 mL borate buffer (0.1 mol. L-1) pH 9.5. After 10 min the absorbance of this solution was measured at 340 nm. A standard curve was prepared using L-isoleucine (0–2 mg. mL-1). DH values were calculated using Eqs 1 and 2.
DH % ¼

n¼

100n
N

DABS  M  d
εP

ð1Þ

ð2Þ

Where N is the the total number of peptide bonds per protein molecule and n is the average
number of peptide bonds hydrolyzed. The ΔABS is the difference in the absorbance of the
hydrolyzed and unhydrolized whey. The component d is the dilution factor, ε is the molar
extinction coefficient (mol. L-1. cm-1) and P the protein concentration of the sample (g. L-1).
The average molecular mass of proteins in the whey (M) was 26.6 kDa and the peptide bonds
per protein molecule (N) was 204.
For the TNBS assay the method used was modified from Spadaro et al. [36]. In this assay 0.2
mL of sample or standard was added to 0.4 mL borate-NaOH buffer (5 mmol. L-1) pH 9.5 and
0.4 mL TNBS (5 mmol. L-1). The mixture was incubated at room temperature for 40 min. The
reaction was stopped by the addition of 0.2 mL 18 mmol. L-1 Na2SO3 prepared in 2 mol. L-1
NaH2PO4 and the absorbance was measured at 420 nm. L-Leucine (0.02–1.5 mmol. L-1) was

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Buffalo Cheese Whey Hydrolysates Production

used as standard and the hydrolysis was calculated using the following formula (Eq 3):
DH ð%Þ ¼ 100

ðAN2 AN1 Þ
Npb

ð3Þ

Where AN1 is the amino nitrogen content of the protein substrate before hydrolysis (mg. g-1)
and AN2 is the amino nitrogen content of the protein substrate after hydrolysis (mg. g-1). The
nitrogen content of the peptide bonds in the whey (Npb) was 123.3 mg. g-1 [37]. The values of
AN were obtained using the following formula (Eq 4):
AN ¼

ABS1
ðε  PÞ

ð4Þ

Where ABS is the absorbance of the sample, ε is the molar extinction coefficient for L-Leucine (mg. L-1) and P is the protein concentration of the sample (g. L-1).

Simulation of the in vitro gastric and intestinal enzymatic digestion
(determination of the dialyzability)
The simulation of gastric and intestinal enzymatic digestion was performed according to Luten
et al. [38]. In this procedure, 95 mL of integral proteins (non-hydrolyzed whey-NH) and
hydrolysates H1 and H2 were adjusted to pH 2.0 with 6 mol. L-1 HCl, followed by the addition
of 2 mL of Milli-Q water and 3 mL of pepsin (1.98x105 U in 0.1 mol. L-1 HCl) at 37°C for 2 h,
simulating gastric digestion. Then the mixture was placed at 4°C for 10 min to minimize pepsin
activity. The peptic digest sample was separated into three aliquots (20 mL each); the aliquots
were transferred to beakers containing cellulose membranes filled with 0.5 mol. L-1 NaHCO3 at
37°C for the dialysis of molecules with molar masses smaller than 12.4 kDa. When pH 7.0 was
reached, 5 mL of 0.1 mol. L-1 NaHCO3 containing 20 mg of pancreatin + 125 mg of bile salts
were added, and the mixture was kept in contact with the membrane for 2 h, simulating intestinal digestion. For determination of the amount of NaHCO3 required to simulate intestinal pH
two aliquots (20 mL each) of the peptic digest were separated to determine the titratable acidity
using 0.5 mol. L-1 NaOH until reaching pH 7.5 and 0.5 mol. L-1 of gram-equivalent.

Peptides and amino acids from the hydrolysis products and enzymatic
digest analyses
H1 and H2 hydrolysates and the dialysates from the dialyzability experiments (solutions inside
the membrane) were filtered using a GV Millex 0.45 μm unit (Millipore) and analyzed by
HPLC (Varian ProStar or Shimadzu LC 10A), which was equipped with a reverse phase column Nucleosil C18 (25 x 0.46 inch, 5 μm particle size, 300 Å pore size). The eluents of the
mobile phase were as follows: A) water and 0.045% trifluoracetic acid (TFA) and B) acetonitrile
(ACN) and 0.036% TFA, with a 5–95% linear gradient of eluent B, a flow rate of 1.0 mL. min-1
for 30 min and with UV detection at 220 nm.
Analysis of the released amino acids was performed using a LC-10A/C-47A Shimadzu automatic analyzer with a fluorescence detector, which was equipped with a Shimadzu Shim-pack
ion-exchange column with o-phtalaldehyde (OPA) post-column functionalization of the
amino acids. Mobile phase A consisted of 19.6 g sodium citrate, 140 mL ethanol 99.5%, 16.7
mL perchloric acid 60%, with a final 1 L volume at pH 10; mobile phase B was 58.8 g sodium
citrate, 12.4 g boric acid, 30 mL 4.0 mol. L-1 sodium hydroxide solution, with a final 1 L volume
at pH 10; mobile phase C consisted of 0.2 mol. L-1 sodium hydroxide solution at pH 13.5, with
a flow rate of 0.6 mL. min-1. A standard amino acid mixture was used to calibrate the system

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Buffalo Cheese Whey Hydrolysates Production

and to provide the elution time of each amino acid and the conversion factor between the peak
area and the concentration of each sample. The molar ratio of amino acids was established,
assuming the concentration of the amino acid closest to the mean for all residues as one unit.

Results and Discussion
Protein, lactose and fat levels in buffalo cheese whey
The protein concentrations were obtained after dialysis, which removed interfering components, such as oligopeptides and amino acid residues released during the enzymatic coagulation
of milk. Lactose and fat concentrations decreased by 98.5% and by more than 90%, respectively,
after dialysis and filtration with kaolin (Table 1). The removal of these compounds is important
for determination of protein concentration with as little as possible interfering.
Results reported by Romám et al. [39] for bovine cheese whey showed lower protein and lactose concentrations compared with buffalo cheese whey, with the exception of the fat concentration. These different protein, lactose and fat levels are most likely related to the analytical
methods used and to the fact that the samples are from different species of mammals and,
therefore, have different environmental and genetic factors that are involved, as described in
the literature [40–41]. The results show that buffalo whey can be considered an excellent source
of high biological value protein that can be used in the food industry.

Comparison between cheese whey proteins from buffalo and bovine by
PAGE and by SDS-PAGE
Ten percent PAGE of bovine and buffalo cheese whey proteins shows a slight difference in the
migration of the protein bands equivalent to bovine serum albumin (BSA) and α-lactalbumin
(α-La), as shown in Fig 1A. The PAGE also shows an extra protein (variant) band in the buffalo
cheese whey in the region between the α-La and β-Lg fractions. There are few published data
concerning the protein profile of buffalo cheese whey; however, a recent study performed by
Buffonni et al. [25] using RP-HPLC showed the presence of two protein peaks corresponding
to α-La. In another study, Chianese et al. [23] used isoelectric focusing showed that α-La has
two genetic variants (α-La A and α-La B).
Twelve percent SDS-PAGE confirmed the presence of a protein band in buffalo whey with
molecular weight ranging from 20.1 to 30.0 kDa and with relative electrophoretic mobility (Rf)
of 0.73 (Fig 1B) that is absent in bovine whey. The determined molar mass of the variant protein was confirmed to be 24kDa by the Least Square Method, determining the molar mass logarithm using the protein Rf (0.73) and, subsequently, the molar mass (Fig 2).
Regarding other proteins, cheese whey from buffalo milk is highly similar to bovine cheese
whey, having proteins with molecular weights between 97 and 66 kDa (immunoglobulins and
serum albumin, respectively) and between 20.1 and 14.4 kDa (β-lactoglobulin and αTable 1. Protein, lactose and fat concentrations of the in natura and treated buffalo milk whey.
Conditions

Protein (g. L-1)

Lactose (g. L1)

Fat (%)

Integral in natura whey

11.13±0.13

65.30±2.20

0.90±0.17

Dialyzed whey*

7.33±0.15

1.23±0.14

0.60±0.10

Treated whey**

6.53±0.49

1.00±0.14

< 0.10

5.40

42.60

2.00

Bovine milk whey (Romám, et al. 2011)
*reduction of lactose
**dialyzed, defatted and centrifuged
doi:10.1371/journal.pone.0139550.t001

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Buffalo Cheese Whey Hydrolysates Production

Fig 2. Determination of the variant protein molecular weight by Least Square Method. Calibration curve
for the determination of the toxin molecular weight by SDS-PAGE (12%). Marker proteins used for calibration
were phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
(30 kDa), trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa). (●) calibration curve; () variant protein.
doi:10.1371/journal.pone.0139550.g002

lactalbumin, respectively). A protein with molecular mass of approximately 30 kDa is also present in both species; however, this protein is most obvious in bovine whey.

Mass spectrometry analysis
Our results indicate the presence of a variant protein in the buffalo cheese whey, which can be
different in the various breeds. According to the MS/MS profiles, five tryptic peptides were
obtained from the 24 kDa protein. These peptides were aligned with the β-Lg sequence from
Bos taurus with 37% of total coverage and high scores (Fig 3). On the other hand, another peptide (VGINYWLAHK) with 7% of coverage for α-La was also obtained with a reduced score.
The peptide data associated to the difference found in the molecular masses (SDS-PAGE and
data bank) indicates the existence of a variant β-Lg in the buffalo cheese whey. Similarly, Chianese et al. [23] found two variants for 0078-La (α-La A and α-La B). These observations reinforce the necessity of new studies concerning this field to clarify the function and importance
of these new proteins.

Determination of the DH for buffalo cheese whey hydrolysates
The TNBS reacts specifically with primary amino groups to form a trinitrophenyl (TNP) derivative that can be quantified using a colorimetric method [42]. However, this method is

Fig 3. Amino acid sequence for β-Lg from Bos taurus (gi/229460). The tryptic peptides obtained for 24 kDa protein from buffalo cheese way were
identical to the red sequence. It was obtained 37% of coverage.
doi:10.1371/journal.pone.0139550.g003
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Buffalo Cheese Whey Hydrolysates Production

Fig 4. Determination of the hydrolysis degree for buffalo cheese whey hydrolysates. The values of DH
were obtained by TNBS and OPA methods for hydrolysis using the M1 and M2 methods.
doi:10.1371/journal.pone.0139550.g004

frequently used to quantify the extension of reactions between reducing sugars and free amino
groups of proteins (Maillard reaction), where a condensation takes place involving mainly the
lysine ε-NH2 and also the terminal α-NH2 [43]. In this sense, the reaction between TNBS and
lysine ε-NH2 is a wide known process, responsible for a background that increases the values
of the DH. A comparative study of the TNP-derivatives absorbance was carry out by Sashidhar
and collaborators [44], where the derivative TNP-lysine had twice the absorbance of the TNPglutamic acid, showing that TNBS reacts equally with both α-amino and ε-amino groups of
amino acids. This effect was observed in determination of the DH for the buffalo cheese whey
hydrolysates (Fig 4), where the values obtained with TNBS assay were higher by a factor of 2
and 1.44, for H1 and H2 respectively.
Another method is based on the specific reaction between OPA and primary amino groups
in the presence of a thiol, forming isoindoles 1-alkylthio-2-alkyl-substituted that can be quantified at 340 nm [35]. The N-acetyl-L-cysteine can be used as thiol in this assay; however, a side
reaction between NAC and cysteine residues can underestimate the DH in whey protein hydrolysates because the cysteine residues compete with NAC for position 1 in the isoindole. Once
this cysteine residue is linked its quantification is not possible, since the peptides and amino
acids of the sample should interact with OPA using the primary amino groups instead of thiol
groups [45]. The determination of the DH for buffalo cheese whey hydrolysates using OPA
presented lower values comparing with TNBS, being 27 and 43% for H1 and H2, respectively.

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Buffalo Cheese Whey Hydrolysates Production

Fig 5. SDS-PAGE patterns for the H1 and H2 hydrolysates. In the both figures the Lane 1: molecular mass standards; Lane 2: treated bovine milk whey;
Lane 3: treated buffalo milk whey. The lanes 4–12 are showing hydrolysates produced using the M1 method (Fig 5a) with incubation between 0–180 min,
whereas the lanes 4–16 are showing hydrolysates produced using the M2 method (Fig 5b) with incubation between 0–1440 min.
doi:10.1371/journal.pone.0139550.g005

SDS-PAGE and HPLC of hydrolysis products
The profile of H1 at different reaction times is showed in Fig 5A. The protein profiles of bovine
and buffalo non-hydrolyzed cheese whey proteins (lanes 2–3), were used to compare the
hydrolysis development by the enzymes pepsin, trypsin, chymotrypsin and carboxypeptidaseA. The disappearance of the variant protein band present in the buffalo cheese whey (lanes
4–5), and the albumin (buffalo whey), indicates its hydrolysis by pepsin during the first 20 min
of the reaction at pH 3.0 and at 50°C. A gradual decrease in the color intensity of the protein
bands clearly indicates the efficiency of the hydrolysis by M1 method (lanes 6 to 12). These
results also show the resistance of α-La and β-Lg to hydrolysis after 180 min under the chosen
experimental conditions (pH 9.5 and 50°C). The SDS-PAGE of the sample with a high degree

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Buffalo Cheese Whey Hydrolysates Production

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Buffalo Cheese Whey Hydrolysates Production

Fig 6. The HPLC chromatographic of the buffalo cheese whey hydrolysates. a) non-hydrolyzed (NH), b)
with a medium degree of hydrolysis (H1), and c) with a high degree of hydrolysis (H2). Reverse phase
chromatography using column Kromasil C18 (250 x 4.6 mm) Φ = 5 μm, 300 Ǻ porosity with a 5–95% linear
gradient (solvent A: water with 0.045% TFA and solvent B: acetonitrile containing 0.036% TFA, 30 min), a
flow rate of 1.0 mL min-1 and with detection at 220 nm.
doi:10.1371/journal.pone.0139550.g006

of hydrolysis (H2) at different reaction times is shown in Fig 5B. Lanes 1 to 4 are the same as in
Fig 5A (same hydrolysis conditions). When using the M2 method, we also observed that the
bulk of proteins in the buffalo cheese whey were hydrolyzed after 24 hours of reaction (lanes
9–16), whereas α-La and β-Lg showed some resistance to the hydrolysis (lanes 5–8).
The non-hydrolyzed (NH) buffalo cheese whey proteins were analyzed by HPLC (Fig 6A),
presenting few peaks (tR 17.0–18.4 min) as expected, because the proteins present in the buffalo
whey were intact in relation to their polypeptide chains. These peaks were reduced with the
enzymatic hydrolysis, and the number of peaks considerably increased after 180 min of hydrolysis for M1 method (Fig 6B), indicating the release of peptides (tR 7.0–16.1 min); however, the
hydrolysis using the M2 method (Fig 6C) was more efficient, because some peptides detected
disappeared after 24 hours of reaction, increasing the release of amino acids. The HPLC results
for the final hydrolysis products of M1 and M2 confirmed the SDS-PAGE results.

Antioxidant capacity
Nowadays, the antioxidant activity of natural products has been studied, among them compounds originating from fruits and vegetables. It has also been known that peptides released
during the enzymatic hydrolysis of proteins are able to modulate certain specific biological
functions such as anti-hypertensive activity, opioids, antimicrobial and antioxidant. Cheese
whey proteins and their hydrolysates (peptides) are potential sources of compounds with antioxidant activity [46–48].
Enzymatic digestion of cheese whey proteins enables the disruption of tertiary structure of
the proteins making the most exposed and accessible amino acid residues to react with free radicals [49]. It is known that cysteine (Cys) is an amino acid that has strong antioxidant activity,
being able to donate hydrogen of its thiol group [50]. Furthermore, Cys is essential for the synthesis of glutathione, an important intracellular antioxidant [18]. However, other amino acid
residues as Tyr, Trp, Met, Phe, His, Ile, Leu and Pro are also able to scavenge free radicals and
act as antioxidants [47,51].
In this study hydrolysates prepared using M2 method showed antioxidant activity for the both
H2 (first hydrolysis) and H2Dint (after gastric and intestinal digestion—dialyzability). These samples showed 4.9 and 12 mmol. L-1 Trolox equivalents, respectively, being able to sequester DPPH•.
When a higher degree of hydrolysis was achieved, as shown in Table 2, there was a release of
amino acids such as Met, Leu, Phe, His, Pro, Tyr, that have the capacity to scavenge free radicals, as previously reported in other studies [47,51].
Another study [52] evaluated the antioxidant activity of free amino acids, showing in vitro
that Tyr and Met have high antioxidant character. Thus, a higher antioxidant activity found in
the sample H2Dint is possibly due to the presence of higher concentrations of Tyr, Phe, Leu
and Val. In addition, HPLC analysis of the sample H2 showed the presence of several peaks
which correspond to peptide bonds, releasing peptides (Fig 6C), which may also be contributing to the antioxidant activity obtained.
[53]. The antioxidant activity of whey proteins hydrolysates depends directly on the molar
mass of the peptide, being the smallest ones (0.1–2.8 kDa) those with strong ability to scavenge
free radicals.

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PLOS ONE | DOI:10.1371/journal.pone.0139550 October 14, 2015

Table 2. Relative concentration (nmol. L-1) of the amino acids from buffalo milk whey before and after dialyzability.
Hydrolysis 11

Dialyzability
NH

H1
2

H2
GD

DExt.

DInt.

Σ AA2

1721.944

426.081

1134.739

398.165

1532.904

902.095

248.826

534.503

191.458

725.961

68.692

256.257

273.693

151.407

75.154

226.561

330.304

0

330.304

0

0

0

0

1242.176

1486.901

1141.124

2628.025

1295.509

1822.587

1050.825

2873.412

1042.983

538.699

659.497

462.148

1121.645

674.240

648.039

484.046

1132.085

712.754

1081.911

302.686

60.797

363.483

1170.781

236.211

309.935

546.146

834.458

2189.711

237.900

1168.512

693.039

1861.551

301.897

1212.762

725.096

1937.858

156.505

36.537

193.042

451.747

199.156

111.345

310.501

514.480

138.848

96.877

235.725

0

0

0

222.233

0

0

0

212.067

0

0

0

260.957

73.925

334.882

623.413

597.372

237.544

834.916

691.035

487.047

251.237

738.284

0

49.514

152.670

31.927

0

31.927

119.370

284.108

350.020

165.579

120.686

286.265

229.531

856.133

531.357

579.769

243.989

823.758

0

0

0

0

0

0

482.645

1215.846

624.271

738.280

485.299

1223.579

Σ AA

GD

DExt.

DInt.

Σ AA

389.537

389.537

386.135

1298.012

423.932

240.016

1368.867

228.764

685.277

216.818

431.575

110.527

542.102

240.577

187.565

0

0

0

0

0

783.473

221.215

0

1270.930

1270.930

319.426

325.491

326.258

586.791

456.192

184.623

604.147

592.122

486.849

225.905

0

392.204

368.867

103.645

1355.253

Asp4

0

25.692

42.094

41.356

Ser4

0

0

0

24.198

Glu4

0

0

0

77.682

Pro4

0

1751.295

2306.616

0

21.802

0

21.802

55.303

49.514

Gly4

0

65.190

87.162

31.687

164.689

37.076

201.765

299.986

164.738

Ala4

0

0

0

40.558

1172.120

133.314

1305.434

474.570

626.602

Cys4

0

0

0

0

333.820

0

333.820

0

0

Tyr4

0

0

0

229.201

541.631

388.603

930.234

669.422

733.201

Amino acid

NH

H1

H2

GD

DExt.

DInt.

Thr3

0

0

0

Val3

0

0

0

50.561

0

32.910

1128.851

Met3

0

94.972

107.605

151.603

Ile3

0

0

201.231

Leu3

0

792.580

Phe3

0

His3

0

Lys3

2

Arg4

0

10.634

6.793

15.681

421.154

251.463

672.617

41.826

514.483

301.067

815.550

54.350

504.479

302.606

807.085

Total

0

3636.616

4833.479

1938.677

7061.997

4448.483

11510.480

6794.662

9003.820

4548.052

13551.872

7521.277

8386.177

4735.373

13121.550

1 Hydrolysis with pepsin, trypsin, chymotrypsin and carboxypeptidase-A
Dext = external intestinal digest, samples collected inside of the membrane; Dint = internal intestinal digest, samples collected outside of the membrane
3 Essential amino acids
4 Non-essential amino acids
doi:10.1371/journal.pone.0139550.t002

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Buffalo Cheese Whey Hydrolysates Production

2 Total of released amino acids by dialyzability (Dext + Dint) NH = non-hydrolyzed; H1 = low degree of hydrolysis; H2 = high degree of hydrolysis; GD = gastric digest;

Buffalo Cheese Whey Hydrolysates Production

Amino acids released before and after gastric and intestinal enzymatic
digestion
A greater variety of amino acids was obtained after gastric and intestinal enzymatic digestion
when compared with NH, H1 and H2 samples (Table 2). M1 method released nine types of
amino acids in the H1 sample, five of which are essential (methionine, leucine, phenylalanine,
histidine and lysine), whereas ten types were detected in the H2 sample, six of which are essential (methionine, leucine, isoleucine, phenylalanine, histidine and lysine), as shown in Table 2.
These results confirmed that amino acids and many peptides were released after enzymatic
hydrolysis for H1 and H2 samples in relation to NH (Fig 6B and 6C).
Samples NH, H1 and H2 subjected to in vitro gastric and intestinal enzymatic digestion
showed a 593.7% increase in the concentration of amino acids released for the NH sample
when compared with gastric digestion only (pepsin action), whereas the increases for H1 and
H2 samples were approximately 99% and 75%, respectively.
Under the study conditions and after a 2h dialyzability period, we demonstrated that diffusion through the semipermeable membrane was 38.6% for the NHDint, 33.6% for the H1Dint
and 36.1% for the H2Dint.
This work demonstrated that the in vitro digestion of integral buffalo cheese whey proteins
(NH) was effective in releasing at least 14 amino acids. Seven of the released amino acids are
essential; the highest concentrations were of valine, leucine, phenylalanine and lysine, which
were diffused through the membrane in the percentages 17.5%, 38.1%, 43.7% and 100%,
respectively.
Tyrosine and arginine also showed significant diffusion rates (41.8% and 37.4%, respectively). The diffusion rates of the other amino acids were below 32%, showing that enzymatic
digestion was effective in releasing essential amino acids and that buffalo cheese whey may
become an important source of these compounds. Notably, the detection of amino acids by the
method employed directly depends on their net charges, which change according to test conditions due to the different pH ranges employed.
Residues of isoleucine, tryptophan, asparagine and glutamine were not detected in any of
the digestion steps.
Due to the prior hydrolysis of H1 and H2, a higher concentration of free amino acids was
released and several peptides were formed. After the enzymatic digestion of both H1 and H2
samples, at least 14 of the twenty amino acids directly involved in bodily functions were
released and detected after dialyzability; most of these amino acids are essential for mammals,
including humans, and must be included in the diet. Among the essential amino acids, leucine
was released in higher concentrations from H1 and H2, with diffusions of 43.4% and 36.6%,
respectively. Leucine is involved in important processes, such as synthesizing and degrading
muscle proteins and in stimulating the release of insulin from the pancreas. Thus, leucine is
already being seen as a pharmaconutrient of great relevance for the supplementary feeding of
malnourished and frail elderly and for specific subpopulations. In the case of the elderly, leucine could minimize sarcopenia, particularly in patients with type 2 diabetes due to its insulinotropic properties. In addition, leucine can also aid in muscle protein synthesis because there is
an accelerated decline of muscle mass in these people [54–55]. Other amino acids, such as
lysine, threonine, tyrosine and phenylalanine, were also released in high concentrations by the
enzymatic digestion of the H1 and H2 hydrolysates. The percentage of diffusion in both cases
was approximately 37% for lysine, 25 to 26% for threonine, 40% for tyrosine and 41 to 42% for
phenylalanine, demonstrating that greater diffusion is not directly related to increased release
because dialyzability is an in vitro method that provides us only a preliminary result that
requires further in vivo studies. The remaining released amino acids, even at lower

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Buffalo Cheese Whey Hydrolysates Production

concentrations and diffusion percentages, are involved in regulating processes such as gene
expression. In vitro studies indicate that dietary supplementation with glutamine and arginine
increased the expression of genes with antioxidant properties, reducing the expression of proinflammatory genes. Another regulatory function attributed to amino acids is the synthesis
and secretion of hormones, such as tyrosine and phenylalanine, which are precursors of the
synthesis of epinephrine, norepinephrine, dopamine and thyroid hormones [56]. There is also
a well-established immunological function related to the amino acids glutamine and arginine,
methionine and cysteine, among others [56–57].
The hydrolysis of the proteins before gastrointestinal digestion favored the greater qualitative
and quantitative release of amino acids. This finding was also observed in recent studies performed in our laboratory with bovine milk whey [26]. In relation to this same work, the amount
of released amino acids from buffalo milk whey is much higher than the amount released from
bovine milk whey. Our findings are interesting from a nutritional perspective because the use of
whole proteins as a supplement produces the same effect as a pre-hydrolyzed supplement.

Conclusions
In this study the protein profile of the buffalo cheese whey was analyzed in comparison with
the bovine milk whey. The profiles are highly similar, except for one protein that is not present
in the bovine milk whey. This protein, with 24 kDa, was analyzed by mass spectrometry, and
the obtained peptides showed greater homology for β-lactoglobulin, being possibly a variant of
this protein. Enzymatic hydrolysis, followed by in vitro gastrointestinal digestion, increased the
release of amino acids, most of which essential amino acids, in higher concentrations when
compared with bovine milk whey and able to scavenge free radicals. Buffalo cheese whey was
found to be a successful alternative source of essential amino acids while presenting high biological value proteins. Moreover, these amino acids are bioavailable to perform their respective
physiological functions.

Acknowledgments
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (J. C. Bassan), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (T. M. S. Bezerra) and Programa Nacional de Pós-Doutorado (PNPD-CAPES) (A. J. Goulart) for financial and research
support. Rubens Monti is a Research Fellow of CNPq. The authors also thank Programa de
Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas (PADC-FCFUNESP), Búfalo Dourado (Fazenda Santa Elisa, Dourado, SP, Brazil) for the donation of the
buffalo milk, and André Roberto Bassan for molecular mass calculus using Least Square
Method.

Author Contributions
Conceived and designed the experiments: JCB AJG ALMN TMSB SSG CBR LHSG RM. Performed the experiments: JCB ALMN TMSB SSG CBR LHSG RM. Analyzed the data: JCB AJG
ALMN TMSB SSG CBR LHSG RM. Contributed reagents/materials/analysis tools: RM CBR
LHSG SSG. Wrote the paper: JCB AJG TMSB RM.

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